1. Field of the Invention
The present invention relates to the efficient commercial synthesis of 1-glycosyl-5-azacytosines, hereafter referred to as 5-azacytosine nucleosides. The inventive methods are particularly useful in preparing 5-azacytosine nucleosides such as 5-azacytidine (azacitidine), 2′-deoxy-5-azacytidine(decitabine). It is well known that azacitidine and its ribodeoxy derivative decitabine are useful in the treatment of disease, especially for myelodysplastic syndrome (MDS).
2. Description of the Related Arts
Examples of 5-azacytosine nucleosides and their syntheses have previously been reported. Azacitidine (also known as 5-azacytidine, 5-AC and Vidaza™) and its ribodeoxy derivative decitabine (also known as 2′-deoxy-5-azacytidine, 5-aza-2′-deoxycytidine, DAC, Dacogen®) were first synthesized as potential chemotherapeutic agents for cancer. A number of methods have been developed to make them but these methods, on the whole, are inefficient and less desirable for commercial production. One important problem is that when the 5-azacytosine ring (s-triazine ring) is conjugated to a carbohydrate, it is sensitive to decomposition by water (under neutral, basic and acidic conditions) and in fact undergoes facile hydrolysis in aqueous formulations, in aqueous emulsions, in aqueous solutions and when exposed to moisture in aqueous work-up during synthesis making commercial manufacture challenging.[1],[13] Therefore it is desirable to develop a production process which limits or avoids the contact of these nucleosides with water.
See, e.g., the following references:    (1) J. A. Beisler, J. Med. Chem., 1978, 21, 204.    (2) U.S. Pat. No. 3,350,388 (1967) and DE1922702 (1969), {hacek over (S)}orm and Pískala (Ceskosl Ovenska Akademieved); A. Pískala and F. {hacek over (S)}orm, Collect. Czech. Chem. Commun. 1964, 29, 2060.    (3) M. W. Winkley and R. K. Robins, J. Org. Chem., 1970, 35, 491.    (4) A. Pískala and F. {hacek over (S)}orm, Nucl. Acid Chem., 1978, 1, 435.    (5) DE2012888 (1971), Vorbrüggen and Niedballa (Schering A G).    (6) U. Niedballa and H. Vorbrüggen, J. Org. Chem., 1974, 39, 3672-3674.    (7) U.S. Pat. No. 7,038,038 (2006), Ionescu and Blumbergs (Pharmion Corporation).    (8) H. Vorbrüggen, K. Krolikiewicz and B. Bennua, Chem. Ber., 1981, 114, 1234-1255.    (9) U.S. Pat. No. 4,082,911 (1978), Vorbrüggen (Schering Aktiengesellschaft).    (10) U.S. Pat. No. 6,887,855 (2005), Ionescu and Blumbergs and Silvey (Pharmion Corporation, Ash Stevens, Inc.).    (11) U.S. Pat. No. 6,943,249 (2005), Ionescu and Blumbergs and Silvey (Ash Stevens, Inc., Pharmion Corporation).    (12) J. Ben-Hatter and J. Jiricny, J. Org. Chem., 1986, 51, 3211-3213.    (13) L. D. Kissinger and N. L. Stemm, J. Chromatography, 1986, 353, 309-318.    (14) U. Niedballa and H. Vorbrüggen, J. Org. Chem., 1974, 39, 3654-3660.
The entire content of each of the above references is incorporated herein as reference.
Pískala and {hacek over (S)}orm[2] teach a lengthy method for the synthesis of azacitidine and decitabine which involves the use of reactive N-glycosylisocyanate intermediates possessing 1-β-configuration. The synthetic process (Scheme 1) comprises reacting a peracylglycosyl isocyanate with an S-alkylisothiurea to obtain a peracylglycosylisothiourea, condensing the latter with an orthoester of an aliphatic acid at high temperature (135° C.) to obtain hydroxy-protected glycosyl-4-alkylmercapto-2-oxo-1,2-dihydro-1,3,5-triazines followed by deprotection with ammonia (NH3) in methanol (MeOH) in a sealed vessel over a 12-24 hour period. Although based on the isocyanate, the overall yield of azacitidine is 43% and the overall yield of decitabine is 33%, it could be difficult to store the isocyanate and its use might provide a health risk. The route also suffers from other difficult to scale-up steps, including the use of the carcinogenic ICH Class I solvent benzene, and the need for a pressure vessel in the deprotection step.

Another potential process for azacitidine and decitabine was reported by Winkley and Robins[3] (Scheme 2). Their approach utilizes the non-catalysed coupling of 1-halosugars with 2-[(trimethylsilyl)amino]-4-[(trimethylsilyl)oxy]-s-triazine(silyl 5-azacytosine) which probably proceeds via an SN2 mechanism. Pískala and {hacek over (S)}orm[4] also reported a similar process utilizing a 1-chlorosugar for the synthesis of azacitidine (Scheme 2), which suffers from the need for gaseous hydrogen chloride in the synthesis of the 1-chlorosugar, very low overall yields (azacitidine in 11%, and decitabine in 7% overall yield[3]), long reaction times (3-7 days), the need for pressure vessels in the deprotection step, the instability of the halosugars, complicated column chromatography and lengthy work-up and isolation procedures.

Niedballa and Vorbrüggen[5,6] teach the synthesis of protected (blocked) nucleosides including azacitidine and decitabine that utilizes a large amount of tin chloride in dichloroethane (DCE) or acetonitrile (MeCN) to promote the coupling of 5-azacytosine and protected sugar moieties (Scheme 3). According to Ionescu and Blumbergs[7] there are a number of major drawbacks to this process: first, removal of tin from the API is difficult Second, emulsions developed during the workup of the coupling mixture. Third, a difficult filtration step needs to be performed in order to isolate the insoluble tin salts. For these reasons it should be concluded that this process is not suitable for the commercial manufacture of azacitidine.[7].

Vorbrüggen[9] teaches a general method for the coupling of silylated bases and nucleoside bases (including cytosine, pyridines triazoles, and pyrimidines, but not 5-azacytosine) with protected 1-O-acyl, 1-O-alkyl or 1-halosugars (viz., ribose, deoxyribose, arabinose and glucose derivatives) in benzene, DCE or MeCN to make protected nucleosides (Scheme 4). The coupling is promoted by trimethylsilyl (TMS) esters of esterifiable mineral acids or strong sulfonic acids, including trimethylsilyl triflate (TMSOTf), TMSOClO3 and TMSOSO2F. The requirement for an aqueous work-up makes this method less than desirable for synthesis of azacitidine and decitabine.

Ionescu and Blumbergs[7] teach a manufacturing process (Scheme 5) specifically for the synthesis of azacitidine which is based on the general trimethylsilyl ester promoted coupling methodology invented by Vorbrüggen.[9] Silylation of 5-azacytosine is conducted using an excess of hexamethyldisilizane (HMDS) and a catalytic amount of ammonium sulfate. The 1-O-acyl-carbohydrate and silylated nucleoside base coupling reaction is carried out in a “solvent having low water solubility” such as dichloromethane (DCM) in the presence of a greater than stoichiometric amount of the non-metallic Lewis acid catalyst TMSOTf. An aqueous workup is employed and a number of costly steps are necessary to remove harmful water and switch solvents into suitable conditions for the deprotection step.

Therefore, there is a need for a more efficient process for manufacturing a 5-azacytosine nucleoside compound on a large scale.